Patent Application
20220373542
Various viruses pose a great threat to populations around the world and can lead to pandemic when virus spread is uncontrolled. For example, the SARS-CoV-2 virus has recently caused a worldwide pandemic. This virus is very contagious and has spread quickly. The virus is also capable of being transmitted from infected individuals before the individuals experience symptoms, and some individuals may remain asymptomatic. Therefore, preventing the spread of this virus has been difficult. The Zika virus (ZIKV) is another example of a virus that has spread with severe consequences. This virus can spread from a pregnant woman to the fetus to cause birth defects such as microcephaly and other congenital abnormalities. The virus can also cause Guillain-Barré syndrome, neuropathy and myelitis in adults. Early detection of these viruses can be very helpful to control the spread of the viruses and help reduce infection rates. Detection methods for different viruses can vary. Some example detection methods that have been used include serum analysis with viral RNA or antibody-based detection assays, polymerase chain reaction-based (PCR) assays, and others. In many cases, these tests can provide useful results but the tests may require expensive and specialized laboratory equipment and procedures, and some tests can have long wait times before results are available.
The present disclosure is drawn to micro-balance based sensors that can detect whole viruses through a frequency response. In one example, a method of detecting a virus can include exposing a sensor surface to a fluid sample containing a suspected virus. The sensor surface can be a surface of a resonator having a resonant frequency from about 1 MHz to about 1 GHz before mass-loading and viral biomolecule attachment. The resonator can be a piezoelectric resonator (e.g. a quartz crystal microbalance) or it can be a micro-electromechanical resonator. The surface of these resonators can be modified with molecular recognition groups selective for binding to the virus or other viral biomolecules (e.g. antigens, antibodies, etc). A resonant frequency of the resonator can be measured after exposing the sensor surface to the fluid sample. The resonant frequency of these devices changes when mass is added to their surfaces. As such, the change in the measured resonant frequency before and after the virus/pathogen is used to detect and quantify the amount of added mass due to viruses or pathogens.
Additional features and advantages of these principles will be apparent from the following detailed description, which illustrates, by way of example, features of the invention.
These drawings are provided to illustrate various aspects of the invention and are not intended to be limiting of the scope in terms of dimensions, materials, configurations, arrangements or proportions unless otherwise limited by the claims.
Reference will now be made to exemplary embodiments and specific language will be used herein to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended. Alterations and further modifications of the inventive features described herein, and additional applications of the principles of the invention as described herein, are to be considered within the scope of the invention. Further, before particular embodiments are disclosed and described, it is to be understood that this invention is not limited to the particular process and materials disclosed herein as such may vary to some degree. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present invention will be defined only by the appended claims and equivalents thereof.
In describing and claiming the present invention, the following terminology will be used.
The singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a layer” includes reference to one or more of such structures, “a metal” includes reference to one or more of such materials, and “a measuring step” refers to one or more of such steps.
As used herein, “substantial” when used in reference to a quantity or amount of a material, or a specific characteristic thereof, refers to an amount that is sufficient to provide an effect that the material or characteristic was intended to provide. The exact degree of deviation allowable may in some cases depend on the specific context. Similarly, “substantially free of” or the like refers to the lack of an identified element or agent in a composition. Particularly, elements that are identified as being “substantially free of” are either completely absent from the composition, or are included only in amounts which are small enough so as to have no measurable effect on the composition.
As used herein, “about” refers to a degree of deviation based on experimental error typical for the particular property identified. The latitude provided the term “about” will depend on the specific context and particular property. The term “about” is not intended to either expand or limit the degree of equivalents which may otherwise be afforded a particular value. Further, unless otherwise stated, the term “about” shall expressly include “exactly,” consistent with the discussion below regarding ranges and numerical data. However, unless otherwise enunciated, the term “about” generally connotes flexibility of less than 2%, most often less than 1%, and in some cases less than 0.01%.
Concentrations, dimensions, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of about 1 to about 200 should be interpreted to include not only the explicitly recited limits of 1 and 200, but also to include individual sizes such as 2, 3, 4, and sub-ranges such as 10 to 50, 20 to 100, etc.
As used herein, the term “at least one of” is intended to be synonymous with “one or more of.” For example, “at least one of A, B and C” explicitly includes only A, only B, only C, and combinations of each.
As used herein, a plurality of items, structural elements, compositional elements, and/or materials may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, no individual member of such list should be construed as a de facto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary.
The present technology includes a variety of sensors that can detect viral biomolecules based on a unique signature frequency response. The various ways in which frequency responses are used in these sensors are described in more detail below. In some examples, any of the sensors described herein can incorporate aptamers or other molecular recognition groups for selectively binding a target virus.
Viral biomolecules can be any chemical entity which is directly or indirectly related to the presence of viruses by indicating a current or previous infection with the virus. Non-limiting examples of viral biomolecules can include whole virus, antibodies, antigens, viral proteins, viral RNA, viral DNA, viral biomarkers, and the like.
Molecular recognition groups can be any chemical compound or group that selectively binds with the target virus. Non-limiting examples of molecular recognition groups can include aptamers, antigens, antibodies, and the like.
Aptamers are molecules that are capable of selectively binding to specific viruses. In some examples, aptamers can be oligonucleotides that can bind to specific sites on a virus. Aptamers can be highly specific. Unlike antigens, which often deteriorate over time, aptamers can remain viable almost indefinitely at room temperature storage. In some examples, when an aptamer binds to a target, the charge content of the aptamer can shift and the configuration of the aptamer can change. These changes and other changes associated with the aptamer binding to a target can be monitored to detect the binding between the aptamer and the target. Accordingly, when an aptamer that specifically binds to a target virus is used, the presence of the virus can be detected by monitoring binding of the aptamer to the virus.
Aptamers can be made artificially and designed specifically to bind to a particular target. In one example, aptamers for binding to Zika virus can be obtained from BasePair Biotechnologies Inc. Aptamers can be obtained for any target virus using the SELEX process (Sequential Evolution of Ligands by Exponential Enrichment) or other similar processes capable of producing aptamers (e.g. modified SELEX, SELCOS, MAWS, JAWS, etc.). In this process, a large library of oligonucleotides can be tested to find oligonucleotides that bind to a target. The targets can include proteins, peptides, carbohydrates, small molecules, toxins, or cells.
In the sensors described herein, aptamers targeting a specific virus can be immobilized on a sensor surface. Molecular recognition groups can generally include a surface bonding group and a virus or other viral biomolecule binding group. Metal surfaces can be bonded with aptamers, antigens, antibodies, or other molecular recognition groups using any suitable functionalization technique. Further, metal surfaces can optionally be first prepared or activated via functionalization with an active group which binds with a corresponding end of the molecular recognition group. For example, a thiol group can be attached to the metal surface. However, in many cases, the molecular recognition group can include a surface bonding group which directly bonds to the metal surface. Non-limiting examples of surface bonding groups can include organosulfur thiols such as alkyl thiols, dialkyl disulfides, etc. Metal, e.g. gold, surface can also be functionalized via techniques such as, but not limited to, oligonucleotide functionalization via thiol groups, surface saturation with single stranded oligonucleotides, PEGylation optionally including thiol or azide bonding groups, photonic immobilization, azide functionalization, and the like. For some details on the known synthetic functionalization techniques, see INNOVACOAT Gold coatings; Polo E. et al. (2013) Tips for the Functionalization of Nanoparticles with Antibodies. In: Guisan J. (eds) Immobilization of Enzymes and Cells. Methods in Molecular Biology (Methods and Protocols), vol 1051. Humana Press, Totowa, N.J. pp. 149-163; Tiwari et al. Nanomaterials 2011, 1(1), 31-63, Functionalized Gold Nanoparticles and Their Biomedical Applications; which are each incorporated herein by reference. For example, thiols can be functionalized at one or both ends of an aptamer, an antigen, or an antibody.
In a specific example, the aptamers can include a thiol end group configured to bind with gold electrodes. Thiol end groups bind with almost any materials. Thiol end groups are very aggressive and in some cases may cause corrosion of some metallic surfaces. In those cases other functional end groups with lower binding energies can be used such as, but not limited to, metal-carbon (e.g. carbene, acetylide, vinylidene, etc), metal-nitrogen (e.g. nitrene, etc), azides, and the like.
Formation of a layer of molecular recognition groups can be performed using liquid phase deposition. A molecular recognition group (e.g. aptamer) solution can be applied to the surface in order to bond with the surface. The functionalized group can then react with the exposed surface to bind the molecular recognition groups to the surface. Residual unreacted materials can be removed by washing or evaporation.
The sensor surface can then be exposed to a sample fluid that is suspected to contain the virus. Because the viral biomolecules such as aptamers can bind specifically with the target virus or other viral biomolecule, the target viral biomolecule can selectively adhere to the molecular recognition group-coated surface. In some examples, aptamers having a linking group, such as a thiol linking group, can be used. The linking group can bond to the sensor surface. Thiol linking groups can covalently bond to a gold surface or almost any other type of metal surface. Accordingly, in some examples, the sensor surface can include a layer of metal such as gold for the aptamers to bond to. In various examples, a metal layer can be formed on a sensor surface using methods such as photoetching, electroplating, electroless plating, physical vapor deposition, and others.
One method of detecting viruses disclosed herein can involve a resonator that can have a measurable resonant frequency that changes in response to the present of a target virus. In one example, a biosensor can include a piezoelectric resonator having a resonant frequency. A surface of the resonator can be modified with aptamers that are selective for binding to a target virus. When the target virus binds to the aptamer, the resonant frequency of the piezoelectric resonator can change due to the increase in mass from the addition of the virus. Because the aptamers can bind specifically with the target virus and not with other materials, a change in the resonant frequency can accurately indicate the presence of the target virus.
The resonant frequency of the piezoelectric resonator without the virus attached can be defined as the “clean” resonant frequency of the resonator. After exposing the aptamer-modified surface of the sensor to a sample fluid, the resonant frequency can be measured again. In some examples, a sample fluid that is known to contain the target virus can be applied to the sensor surface and the resonant frequency can be measured to determine a resonant frequency that indicates the presence of the target virus bound to the aptamers. This serves as a baseline reference. When the sensor is used to test unknown sample fluids, the measured resonant frequency can be compared to this known resonant frequency to determine whether the target virus is present or not. In some examples, the added mass of the target virus can cause the resonant frequency of the resonator to decrease.
In some cases, using a piezoelectric resonator with a relatively high clean resonant frequency can be useful. It has been found that piezoelectric resonators having a relatively high resonant frequency, such as in the range of about 375 MHz to about 500 MHz, can detect target viruses with high sensitivity, although frequencies from about 1 MHz to 1 GHz can also be used. The sensitivity of these sensors can be defined in units of Hz/ng of virus, meaning the number of Hz in the difference between the clean resonant frequency and the resonant frequency after binding to the target virus. Piezoelectric resonators with resonant frequencies in the above range can have a large change in resonant frequency when target viruses are attached. In some examples, the sensitivity of the sensors can be 100 Hz/ng of virus or more, or 200 Hz/ng of virus or more. In certain examples, the sensitivity can be from about 200 Hz/ng of virus to about 600 Hz/ng of virus.
With this description in mind,
In various examples, the fluid sample can be any fluid that is suspected to contain a target virus. The fluid sample can be a liquid or gas. In certain examples, the fluid sample can be ambient air, exhaled air, bodily fluids such as blood, saliva, mucus, urine, etc, aqueous solutions, liquids containing tissue samples such as trachea samples, skin samples, mucus membrane samples, and others. The fluid samples can be used directly or may be diluted in an appropriate solution prior to application to the sensor. The fluid sample can be applied to the surface of the sensor using any suitable application method, such as dropping a liquid sample with an eyedropper, flowing a liquid sample across the surface using a microfluidic system, submerging the sensor in a liquid sample, exposing the sensor surface to ambient air, blowing a gaseous sample across the sensor surface using a blower, blowing an exhaled breath sample across the sensor surface under breath power, and so on.
The piezoelectric resonator can be a material that can generate mechanical energy in response to an applied electric current. The piezoelectric resonator can also have a resonant frequency in the range of from about 1 MHz to about 1 GHz. In certain examples, the piezoelectric resonator can have a resonant frequency from about 430 MHz to about 440 MHz. In still further examples, the piezoelectric resonator can have a resonant frequency from about 433 MHz to about 434 MHz. In some examples, the piezoelectric resonator can include a lithium niobate (LiNbO3) crystal. There are many piezoelectric crystals including quartz and LiNbO3 that can be used in these devices. A notable example is aluminum nitride. In one example, a lithium niobate crystal can have a resonant frequency of about 433 MHz prior to bonding with the aptamer or other molecular recognition group. The resonant frequency changes and is reduced by mass-loading when viruses bound to the surface of the lithium niobate resonator.
More generally, other quartz crystal resonators can be used, although many quartz crystal resonators have a lower resonant frequency. This type of resonator has been used in quartz crystal microbalances (QCM), which can measure mass loading from a virus binding to a surface of the microbalance. When an alternating electric filed is applied, the inner dipole in the quartz crystal causes mechanical strain producing ultrasonic waves, which results in the crystal vibrating at a resonant frequency. The resonant frequency can change when mass is added to the surface of the crystal. The sensitivity is described by the Sauerbrey equation where the change in mass Δm on a certain area A can be calculated from Δm/A=Δfn(ρqμq)0.5/(−2nfl2), where Δfn is the change in resonant frequency, ρq is the density of quartz and μq is the shear modulus of quartz. AT-cut quartz is often used because of its favorable temperature coefficient (3 ppm/(−10° C. to +40° C.)) and its ability to operate in liquids because of its shear-mode vibration.
In some examples, the resonant frequency can change when viruses bind to aptamers on the surface of the piezoelectric resonator. In many examples, the resonant frequency can decrease when viruses bind to the aptamers. Therefore, in some examples, a decrease in the resonant frequency can indicate the presence of the target virus. In certain examples, a decrease in the resonant frequency that is greater than a certain threshold value can indicate the presence of the target virus. In other examples, a resonant frequency with the virus present on the surface can be pre-measured and this resonant frequency can be used as a threshold to determine when the sensor is exposed to sample fluids that contain the target virus. For example, if the measured resonant frequency of the piezoelectric resonator is below the threshold value, then the result can be considered “positive” for the presence of the target virus.
The methods can also include calculating an amount of the target virus present in the sample fluid or present on the aptamer-modifier surface of the sensor. In some examples, a correlation can be developed that can allow the amount of virus present to be calculated based on the measured resonant frequency of the piezoelectric resonator. As an example, a series of sample fluids having multiple different known concentrations of the target virus can be applied to the sensor surface, and the resonant frequency of the piezoelectric resonator can then be measured. The resonant frequencies can be correlated to the concentration of virus in the sample fluids. In other examples, different methods can be used to measure the amount of target virus that is present on the sensor surface, such as direct observation with a microscope. If the amount of virus bound to the sensor surface is known, then the measured resonant frequency of the piezoelectric resonator can be correlated to the amount of virus. This correlation can then be used in future tests when the sensor is used to detect the target virus in unknown sample fluids.
Measurement of the resonant frequency of the piezoelectric resonator can be accomplished using a network analyzer, such as an Agilent™ 4395A Network Analyzer, available from Agilent (USA). Network analyzers are complex instruments that are capable of applying an alternating current to the piezoelectric resonator and measuring the vibration magnitude of the piezoelectric resonator, among many other functions. In some examples, a network analyzer can be used to measure spectrum data of the piezoelectric resonator across a particular span of frequencies. For example, the network analyzer can be used to measure data over a range that encompasses the clean resonant frequency of the piezoelectric resonator. The range can be, in some examples, from about 1 MHz less than the clean resonant frequency to about 1 MHz above the clean resonant frequency. In other examples, the range can be from about 500 kHz less than the clean resonant frequency to about 500 kHz above the clean resonant frequency. In still other examples, the range can be from about 400 kHz less than the clean resonant frequency to about 400 kHz above the clean resonant frequency. The actual resonant frequency of the piezoelectric resonator can be found from the spectrum data.
The methods of detecting viruses can also include outputting a detection signal. The detection signal can be in a variety of forms, including a signal indicating the presence the target virus, a signal indicating the absence of the target virus, a signal indicating an amount of the target virus that is present, a signal indicating a data point such as a measured resonant frequency of the piezoelectric resonator, or another signal related to the detection of the target virus. In various examples, the signal can be an electronic signal that is receivable by a computer, an auditory signal that can be heard by a human user, a visual signal such as a light or an image or text displayed on a display screen, or another type of signal. In a certain example, the detection signal can be a spectrum showing measured spectrum data for the piezoelectric resonator. A human user with appropriate training can read the spectrum data and compare the spectrum data to a spectrum of a clean piezoelectric resonator or to a spectrum of a resonator with the target virus attached, and the human user can determine based on the comparison whether the target virus was present on the sensor surface or not. In other examples, a computer can compare these types of spectrum data and the computer can determine whether the target virus is present and then the computer can output a signal to a human user to indicate whether the target virus is present or not. Any suitable combination of human and computer or electronic devices can be used in these methods for measuring resonant frequencies, comparing resonant frequencies and/or comparing spectrum data, and output detection signals.
Another example frequency based biosensor 300 is shown in
In yet another alternative, the resonator can be a suspended piezoelectric beam with the sensor surface on at least a portion of the suspended piezoelectric beam. As previously described, the sensor surface can include molecular recognition groups which selectively bind with the viral molecule. In this case, the piezoelectric beam can vibrate at a desired frequency based on a corresponding electrical frequency input. The response resonant frequency of the suspended piezoelectric beam will change depending on whether viral biomolecule is attached to the molecular recognition groups due to the increased mass loading on the beam, e.g. a microelectromechanical microbalance resonator sensor.
The beams can be suspended at one or both ends. These beams can be formed of any suitable piezoelectric material such as, but not limited to, lead zirconate titanate, barium titanate, lead titanate, gallium nitride, zinc oxide, piezoelectric polymers (e.g. polyvinylidene difluoride, etc), composites thereof, and the like. Suitable electric contacts and current can be provided by corresponding AC power source which produces a desired input frequency.
It is to be understood that the above-referenced arrangements are illustrative of the application for the principles of the present invention. Thus, while the present invention has been described above in connection with the exemplary embodiments, numerous modifications and alternative arrangements can be made without departing from the principles and concepts of the invention as set forth in the claims.
A biosensor was assembled to demonstrate the detection of Zika virus by measuring a change in resonant frequency of a piezoelectric resonator. The piezoelectric resonator used in this example was made of lithium niobate (LiNbO3). The lithium niobate resonator was extracted from a commercially available communication filter. The lithium niobate resonator had a clean resonant frequency at about 433 MHz. For comparison, similar sensors were build using quartz crystal resonators having resonant frequencies at 5 MHz and 10 MHz. The resonators were coated with gold and then pretreated by rinsing with acetone, ethanol, and deionized water. The resonators were then dried under a nitrogen stream to remove any contaminants from the surface. The resonant frequencies of the resonators were measured using an Agilent™ 4395A Network Analyzer from Agilent (USA). The network analyzer was connected to a computer running a modified version of Lab VIEW software which extracts the resonant frequency from spectrum data.
The resonators were then coated with aptamers that bind specifically to the SF9 envelope protein of the Zika virus. Other aptamers can be used to detect and sense other viruses and pathogens.
An amount of 2 microliters of the 1 micromolar aptamer solution were dropped onto the surface of the resonators. The resonators with the aptamer solution were then kept in a hydrate container at 80° C. for 10 minutes to allow aptamers to bond to the surface. The excess aptamers were then washed off by dipping in deionized water and the resonators were dried under nitrogen stream. The resonant frequencies of the resonators were measured five times. An additional 2 microliters of the aptamer solution was added and washed and dried, followed by measuring resonant frequency five times, and this process was repeated three times at different areas on the resonators.
After this, 2 microliters of stock inactivated Zika virus, and the Zika virus was allowed to bind with the aptamers for 5 minutes. The excess virus was then washed off by dipping in deionized water and drying under nitrogen stream. The resonant frequency was measured again. The Zika virus solution had a concentration of TCID50/mL titer, and the total number of Zika viruses in the 2 microliters of solution was about 3.5×108.
The spectrum and resonant frequency that were measured for the 5 MHz quartz resonator, the 10 MHz quartz resonator, and the 433 MHz lithium niobate resonator, are shown in
The addition of 2 microliters of Zika virus solution was also repeated three times for the quartz resonators.
Three lithium niobate resonators were made and tested. The addition of aptamers was repeated once for two of the sample lithium niobate resonators. Zika virus was only added one time to two of the lithium niobate resonators. The change in resonant frequency with these additions of aptamer and Zika virus are shown in
All of the sensors were able to detect the addition of Zika virus by exhibiting a reduction in the resonant frequency. However, the lithium niobate sensors were much more sensitive than the quartz sensors. The lithium niobate sensors had a sensitivity of about 370 Hz/ng of Zika virus. In comparison, the 5 MHz quartz sensor had a sensitivity of about 1 Hz/ng of Zika virus. Therefore, the lithium niobate sensor has a sensitivity around 400 times greater.
This application claims priority to U.S. Provisional Application No. 62/926,376 filed Oct. 25, 2019 and U.S. Provisional Application No. 63/021,605 filed May 7, 2020, which are each incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US20/57190 | 10/23/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63021605 | May 2020 | US | |
| 62926376 | Oct 2019 | US |
